Imaging Mass Spectrometry · 2019. 11. 4. · 4 6 F L neck SMM SMM 1.5 Positive LN; ANED 5 39 F L...

transcript

Richard M. Caprioli

Mass Spectrometry Research Center Vanderbilt University

Imaging Mass Spectrometry:

Molecular Mapping Beyond the Microscope

NOTE: Confidential information for private viewing only

Mass spectrum from each pixel

Apply matrix to tissue section

4000 6000 8000 10000 12000

Images at single m/z values integrated over all pixels

Laser ablation of array of x,y coordinate positions (pixels)

0% 100%

Basic Approach of MALDI Imaging MS for Tissue Analysis

Mass Spectrometry Technology

1. Imaging/Profiling: MALDI TOF, TOF/TOF, LTQ, IM QTOF, FTICR ( Bruker Autoflex, Ultraflex II, 9.4T FTICR; Thermo LTQ; Waters Synapt; AB STR )

Laser Detector

Sample Plate

MALDI -TOF MS (linear)

2. Molecular ID: LC/MS/MS; LC or 2-D gel, MALDI MS/MS, IM QTOF (Bruker HCT, UltraflexII; Thermo LTQ; AB 4700; Waters Synapt )

Spatial Resolution 1 µm

Sensitivity (signal intensity, # peaks)

200 µm

Data File Size

Time of Acquisition

Imaging Mass Spectrometry - Tradeoffs Exptl Needs

Mouse Kidney – MALDI Imaging MS at 100 µm spatial resolution, 1 kHz rep rate laser, SA matrix

(a) (b)

Human breast tumor cell line implanted into the tibia of a mouse. Human calcyclin (m/z 10,090) Mouse calcyclin (m/z 9960)

Erin Seeley, Lynn Matrisian

1342 1348 1354 1360 0

IMS IHC

Substance P is predominantly localized to the ventromedial SNr

Malin Andersson, Ariel Deutch

300 504 708 912 1116 1320 Mass (m/z)

m/z 1347.8

IMS 100 %

R-P-K-P-Q-Q-F-F-G-L-M - CONH2

b3 b4Y7

R-P-K-P-Q-Q-F-F-G-L-M - CONH2

b3 b4Y7

in situ

standard

Biological / Medical Research

Effects of Diabetes on the Kidney The Glomerulus

1,000,000 PER KIDNEY

Diabetic Nephropathy Goodpasture’s Disease Alport Syndrome

Kerry Grove, Megan Gessel, Billy Hudson

Human Kidney Glomerulus

10-20 years

NORMAL DIABETIC

DIABETICS High blood sugar

Kerri Grove, Billy Hudson

DAMAGE BY OXIDATIVE PATHWAYS INDUCED BY HYPERGLYCEMIA

High glucose

Glycated protein

Reactivecarbonyl species

Reactive oxygen species

Hydroxyl radical (●OH)

Hypohalous acids (HOCl , HOBr)

Glyoxal (CHO – CHO)

Methylglyoxal (CH3 –CO–CHO)

AGEs (advanced glycation end products)

O2 / Mn+

High glucose

Glycated protein

(ΔM = +162)

Reactive carbonyl species

Reactive oxygen species

Hydroxyl radical (●OH)

Hypohalous acids (HOCl , HOBr)

Glyoxal (CHO – CHO)

Methylglyoxal (CH3 –CO–CHO)

Oxidation of Trp, Met, and Cys side chains

(ΔM = +16)

Chlorination and bromination of protein

amino groups (ΔM = +34, +79)

Carboxyethyllysine

(ΔM = +72)

Carboxymethyllysine (ΔM = +58)

5-Hydro- imidazolone (ΔM = +40)

Carboxymethyllysine (ΔM = +58)

Glucosepane (ΔM = +108)

O2 / Mn+

5-Hydro-5-methyl imidazolone (ΔM = +54)

Tetrahydropyrimidine (ΔM = +144)

MALDI IMS

m/z 4343

m/z 4415

4320 4340 4360 4380 4400 4420 4440 4460 0

∆ 72 Da

MS Images of Kidney in Wild Type and Diabetic Mouse

Green: wild type mouse Red : eNOS -/- db/db diabetic mouse

Fig. 2

FTICR MS - CASI (continuous accumulation of selected ions) to enhance dynamic range

Jeff Spraggins

HDR-MALDI FT-ICR mass spectrometry MALDI FT-ICR mass spectrometry

High Dynamic Range Analysis of a Kidney Tissue Section

HDR MALDI FT-ICR MS • Quad mass: m/z 900 • Isolation window: 20 Da • Laser Shots: 6000 • MS/MS used for IDs

Jeff Spraggins, Kerri Grove

Drug Penetration Into Tissue

Mass spectrum from lung tissue biopsy obtained on a low res MS (LTQ)

Study of Rifampicin Treated Rabbit Infected with TB

H&E stain Matrix

m/z 821.169

Rifampicin m/z 821.401

Accuracy: 3 ppm

Bacterial Lipid* PI(33:1)

m/z 821.521 Accuracy: 2.7 ppm

Lipid PG(40:6)

m/z 821.536 Accuracy: 2.2 ppm

*predicted Mycobacterium tuberculosis lipid from the species-specific LipidMaps Database.

Localization of Rifampicin in granulomas in TB infected rabbit lung 30 mg/kg oral dose daily for 1 week. MS/MS RIF [M-H]-

m/z 322 → 397 + 722

Optical image with granulomas indicated H&E image

MS/MS image of RIF (green) overlaid on optical image

Comparison of MALDI and LC-MS

Lisa Manier, Clifton E. Barry, III

Drug Quantitation in Tissue - Mimetic Tissue Model

Tissue homogenates spiked with

range of drug concentrations Frozen polymer mold Cryosection and mount

adjacent to dosed tissue

Frozen Dosed

Tissue Slide for MALDI IMS

Courtesy Reid Groseclose and Steve Castellino, GSK

Drug Quantitation in Tissue

Courtesy Groseclose and Castellino, GSK

SCH 412348 in rat brain: LC/MS vs. MALDI MS

Amount (ng/ml)

MALDI (R2 = 0.93)

LC/MS (R2 = 0.95)

Michelle Reyzer, Walter Korfmacher (Schering-Plough)

Clinical Applications

Reid Groseclose

Serial Tissues Sections Mounted onto MALDI targets

Antigen Retrieval

In situ Trypsin Digestion

MALDI Imaging /Profiling Mass Spectrometry

Working with Formalin Fixed Paraffin Embedded Tissue

MALDI MS Profiling (Histology Directed Molecular Analysis)

Mass spectra mapped to specific locations in tissue based on histology image

-- Fresh frozen tissue -- Formalin fixed paraffin embedded (in situ tryptic digestion)

Erin Seeley, Rossitza Lazova (Yale, Alirezer Zephir (Harvard))

MS Analysis of Spitzoid Lesions in FFPE Biopsies

Spitzoid Melanoma

Spitz Nevi

945 950 955 960 965 970 975 980 1058 1060 1062 1064 m/z

x3 Spitz

Training set

# Patients

Classification Accuracy (%)

Spitz nevi (SN) 26 100

Spitzoid Malignant Melanoma (SMM)

Classificat ion of Spitzoid Lesions

Validation (test) set

# Patients

Classification Accuracy (%)

Spitz nevi (SN) 30 97

Spitzoid Malignant Melanoma (SMM)

56 SN and 54 SMM from Yale University Spitzoid Neoplasm Repository

Primary Ocular Melanoma

Lesion on neck

Lesion on right arm

Case Study I

17 year old boy with ocular melanoma A few months later presented with two skin lesion Skin lesions clinically diagnosed as metastases

Tissue

Pathology

MS Analyses

(# separate areas) Biopsy Malignant

Melanoma

15/15 Malignant Melanoma

Mass Spectrometry Analysis

Tissue

Clinical

Evaluation

MS Analyses

(# separate areas)

Skin lesion #1 Malignant Melanoma

16/17Spitz nevus

Skin lesion #2 Malignant Melanoma

10/10Spitz nevus

Occular melanoma

Skin lesions

36 year old pregnant woman presents with lesion on upper arm

Excisional biopsy performed and determined to be malignant

Insufficient margins taken for size of lesion

No further treatment during pregnancy

Two months later, the baby was born with multiple nevi

Mother Mass Spectrometry

Malignant Melanoma 29/29 Malignant Melanoma

Skin lesions Baby

Mass Spectrometry

A indeterminate 23/23 Nevus

B indeterminate 9/9 Nevus

# Age Gender Site Histologic Dx MS dx Follow up (y) Clinical Status

1 43 M Back SMM SMM 3.5 Negative LN; ANED

2 23 F L calf SMM SMM 2 Positive LN; ANED

3 28 F Thigh SMM SMM 12 Positive LN 8 years later; ANED

4 6 F L neck SMM SMM 1.5 Positive LN; ANED

5 39 F L post leg SMM SMM 1.5 Positive LN; ANED

6 5 F Buttock SMM SMM 6 Positive LN; ANED

7 29 F R upper back SMM SMM 14 Negative LN: Re-excision; ANED;

8 50 M thorax SMM SMM 3 DOD with lung mets 3 years later

9 43 M back SMM SMM 4 Negative LN; ANED

10 57 F NK SMM SMM 3 Negative LN; ANED

LN – Lymph Node; ANED – Alive, No Evidence of Disease; DOD – Dead of Disease

11 15 F L neck SMM SN 4 Negative LN; ANED

12 6 M Abdomen SMM SN 1 ANED

13 44 F R upper arm SMM SN 7 ANED; 2 other ASN favor SN

14 16 M Back SMM SN 10 Negative LN; ANED

15 55 M R mid back SMM SN 2 ANED

16 40 F R upper arm SMM SN 11 Negative LN; ANED

17 9 M R upper arm SMM SN 14 Negative LN; ANED

18 17 M Chest SMM SN 1 Negative LN; ANED

20 44 F

R buttock SMM SN 9 Negative LN; ANED

R upper arm SMM SN 8 Negative LN; ANED

21 30 F R shin SMM SN 14 ANED

22 57 M R thigh SMM SN 12 ANED

23 46 M R arm SMM SN 4 1 Positive LN-1 cell; ANED

# Age Gender Site Histologic Dx MS dx Follow up (y) Clinical Status

1 43 M Back SMM SMM 3.5 Negative LN; ANED

2 23 F L calf SMM SMM 2 Positive LN; ANED

3 28 F Thigh SMM SMM 12 Positive LN 8 years later; ANED

4 6 F L neck SMM SMM 1.5 Positive LN; ANED

5 39 F L post leg SMM SMM 1.5 Positive LN; ANED

6 5 F Buttock SMM SMM 6 Positive LN; ANED

7 29 F R upper back SMM SMM 14 Negative LN: Re-excision; ANED;

8 50 M thorax SMM SMM 3 DOD with lung mets 3 years later

9 43 M back SMM SMM 4 Negative LN; ANED

10 57 F NK SMM SMM 3 Negative LN; ANED

11 15 F L neck SMM SN 4 Negative LN; ANED

12 6 M Abdomen SMM SN 1 ANED

13 44 F R upper arm SMM SN 7 ANED; 2 other ASN favor SN

14 16 M Back SMM SN 10 Negative LN; ANED

15 55 M R mid back SMM SN 2 ANED

17 9 M R upper arm SMM SN 14 Negative LN; ANED

18 17 M Chest SMM SN 1 Negative LN; ANED

20 44 F

R buttock SMM SN 9 Negative LN; ANED

R upper arm SMM SN 8 Negative LN; ANED

21 30 F R shin SMM SN 14 ANED

22 57 M R thigh SMM SN 12 ANED

23 46 M R arm SMM SN 4 1 Positive LN-1 cell; ANED

LN – Lymph Node; ANED – Alive, No Evidence of Disease; DOD – Dead of Disease

# Age Gender Site Histologic Dx MS dx Follow up (y) Clinical Status 1 35 F L thigh ASN SN 5.5 ANED

2 33 M Back ASN SN 7 ANED

3 14 M L arm ASN SN 7 ANED

4 11 M Posterior neck ASN SN 7 ANED

5 55 F L upper back ASN SN 7 ANED

6 3 M R ear ASN SN 7 ANED

7 16 M R canthus ASN SN 6 ANED

8 34 M R leg ASN SN 1 ANED

9 7 M Scalp ASN SN 5 ANED

10 28 F Breast ASN SN 6 ANED

11 18 M Neck ASN SN 2.5 0/1 SLN; ANED

12 39 F R ankle/foot ASN SN 6 ANED

13 34 F R shin ASN SN 6 ANED

14 72 F L arm ASN SN 6 ANED

15 13 F R medial knee ASN SN 6 ANED

16 22 F L ant thigh ASN SN 6 ANED

17 49 F L medial thigh ASN SN 5 0/1 SLN; 0/1 LN; ANED

18 30 F R thigh

ASN - orig. bx SN

5 ANED ASN - re-exc. SN

19 3 M R cheek ASN SN 5 ANED

20 26 F R thigh ASN SN 5 ANED

21 38 M R cheek ASN SN 5 ANED

22 6 F R medial thigh ASN SN 5 ANED

23 8 M R extensor elbow ASN SN 5 ANED

24 19 F R upper thigh ASN SN 1 ANED

25 45 M R calf ASN SN 1 0/3 SLN; 0/1 LN; ANED

26 58 F L upper back ASN SN 1 ANED

27 29 F L buttock ASN SN 1 ANED

28 9 M R lateral knee ASN SN 6 1+SLN; 0/1 LN; CGH-no abnormalities; ANED

29 16 M L ear ASN SN 8 ANED

30 4 F R thigh ASN SN 8 ANED

31 27 F L shin ASN SN 15 ANED

32 34 M NK ASN SN 14 ANED

33 48 M L flank ASN SN 13 ANED

34 34 F R post forearm ASN SN 11 ANED

35 37 F R ear ASN SN 6 ANED

36 7 F R lower back ASN SN 15 ANED

37 24 F L buttock ASN SN 14 ANED

38 39 M R post arm ASN SN 6 ANED

New Technology Initiatives

High Spatial Resolution Imaging (1-5 μm) Increased Imaging Speed (<< 1 sec/pixel) Multi-Modal Image Fusion 3-D MS Images Increased Sensitivity – Targeted Analyses, Derivatization Ease of Use: Matrix Pre-Coated Targets

MALDI (TOF) IMS using a 5 kHz Nd: YAG continuous laser

m/z 734.4 ( PC 32:0)

m/z 788.5 (PC 36:1)

m/z 806.5 (PC 38:6)

lipid images acquired at a rate of 30 pixels/s Total image time: < 8 min

Jeff Spraggins

High Spatial Resolution Imaging MS

(1 - 5 µm pixel diameter)

Schematic of a Transmission Geometry Instrument

Andrey Zavalin Ken Shriver

Single Cell / Sub-cellular Imaging Pancreas islet section ablated with 30 shots from a 2-3 µm diameter laser spot on target

Andrey Zavalin, Kerri Grove

5400 6400 m/z

m/z 750 PE (18:0p/20:4) m/z 1052 Hex-Sulfo-Hex-Cer-(d16:1/26:0) m/z 906 SulfoHex-Cer (t18:0/24:1)

100 mm

Ion Images of Human Kidney Cortex Resolution: 1 mm laser beam, 2 mm pitch

25 shots/pixel, transmission geometry matrix sublimed DAN

Analysis of Single Mammalian Cells

HEK-293 cells

MS image: m/z 782

Optical image After ablation

RKO cells

MS image: m/z 782 Optical image after ablation

Image resolution: 1 µm beam diameter at 1.5 µm pitch; 349 nm UV laser; rep rate 1 kHz

Erik Todd, Andrey Zavalin

1pixel 25 shots

25 shots

Single Cell Analysis – HEK 293

Transmission geometry TOF MS, Nd:YLF laser (355 nm), single shot

1 µm laser beam

5 µm laser beam

10 µm 10 µm

Image Processing

IMAGE FUSION

TISSUE

imaging MS

microscopy

tagged

microscopy

Low spatial res.

High chemical info

High spatial res.

Low chemical info

…combines best attributes of imaging technologies …can highlight non-obvious relationships with other technologies (e.g., MRI, PET, microscopy, etc.) …is predictive in nature

A page from satellite imaging...

pan-chromatic image - high spatial resolution - little info per pixel

multispectral image - low spatial resolution - a lot of info per pixel

fused image - high spatial resolution - a lot of info per pixel

IMS – Microscopy Fusion

Creating Multi-Modal Images

Raf Van de Plas

Imaged at 100 um

Microscopy 5 µm MS Image at 100 µm

IMAGE FUSION Mass Spectrometry and Microscopy

Raf Van de Plas

Predictive MS image sharpening through fusion with microscopy m/z 18,399 - transverse section of mouse brain

MS Fused Image sharpened to 5 µm

m/z 18,399

3–D Imaging Mass Spectrometry

3D Imaging MS: Correlation with MRI

Erin Seeley Tuhin Sinah John Gore

H&E staining of the mouse pup - progression from superficial sections containing just skin, muscle, and bone through the abdominal organs and central nervous system and back to superficial sections.

Preparation for 3D Image of Whole Mouse Pup 15 µm sections at 210 µm spacing

Erin Seeley, Tuhin Sinha

Erin Seeley, Eric Skaar

Systemic Staph Infection in Mouse

Erin Seeley, Kaitlin Schroeder, Eric Skaar, Kevin Wilson

Systemic Staph Infection in Mouse Kidney m/z 10,165 (Calgranulin A) located in abscesses

Perspectives

Instrumental / Methodology Challenges

Sensitivity - achieve more global coverage ( fraction of proteome now observed) Resolution - better lateral resolution (routine single cell imaging) - higher MS resolution (better resolve isoforms, PTMs, isobars) Mass Range - routinely beyond 50 kd Identification - in situ - fast, simple, accurate Quantitation - reagents and methods - isotope based, relative and absolute Validation - cross-lab (std protocols), cross-platform reproducibility/standardization Availability - single manufacturer must provide entire technology ‘solution’

IMS - Summary

- measures native molecular distributions, providing new biological insights that easily correlate with other imaging modalities - is an excellent discovery technology because no target specific reagents are needed - has exceptionally high throughput (in some cases less than a few seconds for data acquisition per sample), providing multiple images simultaneously at discrete MW values - can undergo fusion with data from other imaging modalities to create new imaging paradigms

Mass Spectrometry Research Center Jeremy Norris Erin Seeley Michelle Reyzer Andrey Zavalin Jeff Spraggins Peggi Angel Lisa Manier Junhai Yang Kerri Grove Raf Van de Plas Megan Gessel David Anderson Brian Hachey Boone Perkins

Vanderbilt Collaborators David Hachey Kevin Schey Paul Laibinis John Gore Charles Manning Eric Skaar Billy Hudson Nancy Brown Randy Blakely Anna Carneiro Ariel Deutch Ray Mernaugh Melinda Sanders Kay Washington Kevin Wilson

Funding NIH GMS – 3D Imaging NCRR/GMS - National Resource for IMS Department of Defense The Gates Foundation Vanderbilt University

Others Peter Wild, U Zurich Reid Groseclose, GlazoSmithKline Kristin Burnum, Batelle PNW Labs John Mayer, Harvard Pierre Chaurand, U Montreal Shannon Cornett, Bruker Daltonics Ron Kahn, Harvard Andre Kleinridders, Harvard SK Dey, U Cincinnati Giovanni Sindona, U Calabria Alireza Sepehr (Harvard) Rossitza Lazova (Yale) Gwendoline Thiery, Harvard Kristina Schwamborn, Univ. Munich

Thank You

Imaging Mass Spectrometry · 2019. 11. 4. · 4 6 F L neck SMM SMM 1.5 Positive LN; ANED 5 39 F L...

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